CN108398423B - Renin chemiluminescence detection kit - Google Patents
Renin chemiluminescence detection kit Download PDFInfo
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Abstract
The invention discloses a renin chemiluminescence detection kit, which comprises a reagent 1 and a reagent 2, wherein the reagent 1 comprises a buffer solution, magnetic particles, a biotin-labeled anti-renin monoclonal antibody and polyhydroxy alcohol; and said reagent 2 comprises a buffer, an acridinium ester-labeled anti-renin monoclonal antibody and a polyhydroxy sugar. The renin assay kit has high sensitivity and good accuracy and can reduce assay cost.
Description
Technical Field
The invention relates to the field of in-vitro diagnostic reagents, in particular to a renin chemiluminescence detection kit.
Background
Renin (English: Renin), also known as angiotensinogenase, is a proteolytic enzyme released by the pericentral cells of the paracoccus renal (also known as the paracoccus complex) apparatus and is a component of the Renin-angiotensin system. Renin enters the blood through the renal vein, catalyzes the conversion of angiotensinogen secreted by the liver into plasma into angiotensin i, and increases the production of aldosterone. Increasing the tubular reabsorption of NaCl and water by convertases in blood and lung tissues degrades angiotensin i to angiotensin ii, which can be hydrolyzed by aminopeptidase to angiotensin iii. The three angiotensins have biological activity, wherein the biological activity of the angiotensins II and III is stronger, and the concentration of the angiotensin II in blood is lower, so the biological activity of the angiotensins II is strongest. Angiotensinogen and convertase, etc., are often present in plasma, and the release of renin is a critical condition in determining the concentration of angiotensin in plasma.
There are currently two methods for clinically detecting renin: renin activity and renin concentration (also known as direct renin approach). Renin Activity (PRA) is a classical method, is used for determining the rate of renin-catalyzed angiotensin I production in a unit blood sample, needs to incubate the blood sample for a period of time first and then determine the concentration of the newly produced angiotensin I, the detection process is relatively complex, manual operation is generally adopted, the operation process is complicated, the consumed time is relatively long, and the error of the detection result caused by human factors is relatively large.
The renin concentration is the quality of the renin molecules in a unit blood sample, generally, two monoclonal antibodies are used for carrying out double-antibody sandwich method detection on the renin molecules, and common detection methods comprise an enzyme-linked immunosorbent assay, a radioimmunoassay and a chemoimmunoassay luminescence method, but the methods have some defects. The enzyme-linked immunosorbent assay is mostly operated manually, and the automation degree is low; the operation steps are complex and long in time; the accuracy and precision of the detection result are poor, and the sensitivity is low. The radioimmunoassay has the disadvantages of large radioactive pollution, complex operation, long experiment time consumption, short kit storage time, unstable detection result and low sensitivity. The chemiluminescence immunoassay method is commonly plate-type chemiluminescence and magnetic particle chemiluminescence. The plate-type chemiluminescence method is similar to the enzyme-linked immunosorbent assay, the automation degree is low, manual operation is mostly adopted, the detection result has larger error caused by human factors, and the sensitivity is lower; the magnetic particle chemiluminescence method adopts a full-automatic instrument for detection, has high automation degree and more stable result, but the existing clinical kit generally has the problems of low sensitivity and insufficient low-end resolution, and adopts multi-point calibrator calibration to increase the reagent detection cost and the burden of patients.
In China, the detection of renin is mainly performed by a chemiluminescence immunoassay in clinic, but as the domestic renin detection kit with independent intellectual property rights is less at present, the sensitivity is lower, the reagent cost is high, the detection kit is generally developed in three hospitals at present and is less developed in hospitals below three, so that the detection technology which has high sensitivity and good accuracy for the detection of renin and can reduce the detection cost is to be developed.
Disclosure of Invention
In order to solve the above problems, the present invention provides a renin chemiluminescence detection kit, which comprises a reagent 1 and a reagent 2, wherein the reagent 1 comprises a buffer solution, magnetic particles, a biotin-labeled anti-renin monoclonal antibody and a polyhydroxy alcohol; and said reagent 2 comprises a buffer, an acridinium ester-labeled anti-renin monoclonal antibody and a polyhydroxy sugar.
In one embodiment, the reagent 1 comprises 0.2mg/mL to 0.6mg/mL magnetic microparticles, 4 μ g/mL to 12 μ g/mL biotin-labeled anti-renin monoclonal antibody, and 5g/L to 50g/L mannitol or sorbitol; and the reagent 2 comprises 2.0-6.0 mu g/mL acridinium ester labeled anti-renin monoclonal antibody and 5-50 g/L trehalose or sucrose.
In one embodiment, the reagent 1 comprises a buffer, 0.2mg/mL to 0.6mg/mL magnetic microparticles, 4 μ g/mL to 12 μ g/mL biotin-labeled anti-renin monoclonal antibody, and 5g/L to 50g/L mannitol or sorbitol; and the reagent 2 comprises buffer solution, 2.0-6.0 mu g/mL acridinium ester labeled anti-renin monoclonal antibody and 5-50 g/L trehalose or sucrose. In some embodiments, it may be 0.3mg/mL to 0.5mg/mL magnetic microparticles; can be a biotin-labeled anti-renin monoclonal antibody of 6 mu g/mL-10 mu g/mL; and 10 g/L-40 g/L mannitol or sorbitol, 20 g/L-30 g/L mannitol or sorbitol.
In some embodiments, the buffer is a PBS buffer with pH 6.0-8.0, 5 mM-100 mM. In some embodiments, the buffer may be a PBS buffer with a pH of 7.0-7.5, 20 mM-80 mM, 20 mM-60 mM, and 20 mM-40 mM.
In some embodiments, the reagent 1 further comprises 1-50 g/L bovine serum albumin, 5-20 mL/L glycerol, 0.05-0.2 mL/L TritonX-100, 10-50 mg/L4-aminoantipyrine, and 0.5-5 mL/L proclin-300. In some embodiments, the serum albumin can be 5 g/L-40 g/L, 10 g/L-30 g/L, 15 g/L-25 g/L; 10mL/L to 15mL/L of glycerol; 0.1-0.2 mL/L TritonX-100, 0.15-0.2 mL/L TritonX-100; 20 mg/L-40 mg/L of 4-aminoantipyrine and 15 mg/L-30 mg/L of 4-aminoantipyrine; and 1mL/L to 5mL/L proclin-300, 2mL/L to 4mL/L proclin-300. All components in the reagent are combined to play a role, so that the background signal of the reagent is effectively reduced, and the sensitivity and the stability of the reagent are improved.
In some embodiments, reagent 1 comprises 0.4mg/mL magnetic microparticles, 8. mu.g/mL biotin-labeled anti-renin monoclonal antibody, 20mM pH7.4PBS buffer, 10g/L bovine serum albumin, 20g/L mannitol or sorbitol, 10mL/L glycerol, 0.1mL/L TritonX-100, 20 mg/L4-aminoantipyrine, and 1mL/L proclin-300.
In some embodiments, the reagent 2 further comprises 5 to 20g/L casein, 5 to 20mL/L glycerol, 0.05 to 0.2mL/L TritonX-100, 0.5 to 5mL/Lproclin-300, and 10 to 50 mg/L4-aminoantipyrine. In some embodiments, the reagent 2 may include 5g/L to 10g/L casein, 10g/L to 15g/L casein, 15g/L to 20g/L casein; 5 mL/L-20 mL/L of glycerol, 5 mL/L-100 mL/L of glycerol and 10 mL/L-20 mL/L of glycerol; 0.05 mL/L-0.2 mL/L TritonX-100, 0.05 mL/L-0.1 mL/L TritonX-100, 0.1 mL/L-0.2 mL/L TritonX-100; 0.5 mL/L-5 mL/L proclin-300, 0.5 mL/L-1 mL/L proclin-300, 1 mL/L-2 mL/L proclin-300, 2 mL/L-4 mL/L proclin-300; and 10mg/L to 20mg/L of 4-aminoantipyrine, 20mg/L to 30mg/L of 4-aminoantipyrine, 30mg/L to 40mg/L of 4-aminoantipyrine, and 40mg/L to 50mg/L of 4-aminoantipyrine. All components in the reagent are combined to play a role, so that the background signal of the reagent is effectively reduced, and the sensitivity and the stability of the reagent are improved.
In some embodiments, reagent 2 comprises 4.0. mu.g/mL acridinium ester-labeled anti-renin monoclonal antibody, 20mM pH7.4PBS buffer solution, 10g/L casein, 10g/L trehalose or sucrose, 10mL/L glycerol, 0.1mL/L TritonX-100, 1mL/L proclin-300, and 20 mg/L4-aminoantipyrine. In some embodiments, the kit further comprises a calibrator comprising PBS buffer, renin antigen, casein, bovine serum albumin, trehalose or sucrose, glycerol, triton x-100, proclin-300, and 4-aminoantipyrine.
In some embodiments, the calibrator comprises 10 μ IU/mL and 100 μ IU/mL renin antigen, 5 mM-100 mM, pH 6.0-8.0 PBS buffer, 5 g/L-20 g/L casein, 20 g/L-100 g/L bovine serum albumin, 5 g/L-50 g/L trehalose or sucrose, 5 mL/L-20 mL/L glycerol, 0.05 mL/L-0.2 mL/L TritonX-100, 0.5 mL/L-5 mL/L proclin-300, and 10 mg/L-50 mg/L4-aminoantipyrine. In some embodiments, it may be from 10g/L to 20g/L casein, from 15g/L to 20g/L casein; 30 g/L-80 g/L bovine serum albumin, 40 g/L-60 g/L bovine serum albumin; 10 g/L-40 g/L of trehalose or sucrose, 20 g/L-30 g/L of trehalose or sucrose; 5 mL/L-10 mL/L of glycerol, 10 mL/L-20 mL/L of glycerol and 15 mL/L-20 mL/L of glycerol; 0.1-0.2 mL/L TritonX-100, 0.1-0.15 mL/L TritonX-100; 1 mL/L-4 mL/L proclin-300, 2 mL/L-4 mL/L proclin-300; and 10mg/L to 40mg/L of 4-aminoantipyrine, 20mg/L to 40mg/L of 4-aminoantipyrine and 30mg/L to 40mg/L of 4-aminoantipyrine.
In some embodiments, the calibrator comprises 20mM pH7.4PBS buffer, 10g/L casein, 50g/L bovine serum albumin, 20g/L trehalose or sucrose, 10mL/L glycerol, 0.1mL/L LTritonX-100, 1mL/L proclin-300, and 20 mg/L4-aminoantipyrine.
In some embodiments, the kit further comprises a chemiluminescent substrate solution comprising solution a and solution B; the A solution is H2O2And the solution B is NaOH solution.
According to the invention, through the selection of each component in the reagent 1 and the reagent 2, the signal background of the kit is greatly reduced in use, and the signal intensity is obviously increased; the sensitivity, accuracy and repeatability of the kit are obviously improved. In particular, the presence of mannitol or sorbitol in reagent 1 and trehalose or sucrose in reagent 2 significantly increases the sensitivity of the kit of the invention.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be further described below with reference to the following embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
EXAMPLE first preparation of avidin-modified magnetic microparticles coated with renin monoclonal antibody (reagent 1)
1. Preparation of biotinylated renin monoclonal antibody
Taking 1.0mg of renin monoclonal antibody, diluting to 0.5mg/mL by using phosphate buffer solution, then adding 13.3 mu L of biotin ester with the concentration of 2.5mM/L, reacting for 30min at room temperature, then adding 20 mu L of Tris solution with the concentration of 1M, reacting for 10min at room temperature, finally dialyzing for 16-24 h by using 20mM of phosphate buffer solution with the pH of 7.4, and taking the dialyzed liquid to obtain the biotinylated renin monoclonal antibody.
2. Biotinylated antibody coated avidin magnetic particles
Taking 50mg of the suspension of the avidin-modified magnetic particles, magnetically separating the supernatant, washing with a linking buffer (20mMPBS, 10g/L BSA, 20g/L mannitol, 10mL/L glycerol, 0.1mL/L TritonX-100, 1mL/L proclin-300, 20 mg/L4-aminoantipyrine) three times, then resuspending with 25mL of ligation buffer, adding 1.0mg of biotinylated renin monoclonal antibody, suspending at room temperature for 30min, magnetically separating the supernatant, washing with ligation buffer once, then resuspending to 25mL, then adding 0.5mL of magnetic particle blocking solution (10 mu M/mL of biotin, 50% glycerol and 50% dimethyl sulfoxide by volume in the blocking solution) to suspend for 30min at room temperature, magnetically separating the supernatant, washing with a connecting buffer solution for three times, then suspending to 125mL, thus obtaining the avidin magnetic particles coated by the renin monoclonal antibody of 0.4 mg/mL.
The method also prepares the avidin magnetic particles coated by the renin monoclonal antibody of 0.2mg/mL and 0.6 mg/mL. And reagent 2 comprising biotin-labeled anti-renin monoclonal antibodies of 4. mu.g/mL, 8. mu.g/mL and 12. mu.g/mL was prepared.
EXAMPLE two preparation of acridinium ester-labeled renin monoclonal antibody (reagent 2)
Diluting 1.0mg of renin monoclonal antibody to 2.0mg/mL by using 20mM phosphate buffer solution, then adding 20 mu L of 10mM/L acridine ester, uniformly mixing the acridine ester at room temperature in the dark for reaction for 30min, then adding 100 mu L of 10mg/mL lysine solution, uniformly mixing the acridine ester and the lysine solution at room temperature in the dark for reaction for 10min, and then carrying out centrifugal desalination and purification by using a desalination column. And collecting the liquid in the centrifuge tube to obtain the acridinium ester labeled renin monoclonal antibody. Then diluting the obtained acridinium ester marked renin monoclonal antibody to the concentration of 4.0 mu g/mL by using a marker diluent to obtain a reagent 2, wherein the marker diluent is a PBS solution with 20mM pH7.4 and contains 10g/L casein, 10g/L trehalose, 10mL/L glycerol, 0.1mL/L LTriton X-100, 1mL/L proclin-300 and 20 mg/L4-aminoantipyrine. In addition, an anti-renin monoclonal antibody comprising 2.0. mu.g/mL and 6.0. mu.g/mL acridinium ester labels was prepared in this manner.
EXAMPLE III renin calibrator preparation
The renin antigen is prepared into the concentration of 10 mu IU/mL and 100 mu IU/mL by using a calibrator diluent, and is subpackaged into 1.0 mL/bottle and then is frozen and dried. RENIN calibrators are traceable to the WHO International Standard RENIN, Human, NIBSC code: 68/356. The calibrator diluent 20mM pH7.4 is PBS solution, containing 10g/L casein, 50g/L bovine serum albumin, 20g/L trehalose, 10mL/L glycerol, 0.1mL/L TritonX-100, proclin-3001 mL/L and 20 mg/L4-aminoantipyrine.
EXAMPLE four Performance testing of the renin assay kit
Detecting with full-automatic chemiluminescence immunoassay analyzer, sequentially adding 50 μ L sample, 25 μ L avidin magnetic particles coated with renin monoclonal antibody, and 50 μ L acridinium ester labeled renin monoclonal antibody, reacting for 30min, magnetically separating, cleaning, and adding luminescence substrate solution A (solution A is 0.10mol/L H)2O2Solution), solution B (solution B is 0.15mol/L NaOH solution) to detect the light emission signal. The chemiluminescent immunoassay analyzer may be, for example, the following instruments of the michael organism model: maccura i3000, maccura i3000L, maccura i3000S, maccura i2000L, maccura i2000S, maccura i1000L, maccura i 1000S. The calibrator information is scanned onto the analyzer through the bar code, and the analyzer automatically calibrates the detection system through the calibrator information read by scanning. 1. Sensitivity (minimum detection limit) test of kit
Reagent sensitivity was determined based on the lowest limit of detection (LOB) which was performed as described below. Detecting the zero concentration calibrator 20 times to obtain a signal value (RLU) of 20 measurement results, calculating the average value M and standard deviation SD to obtain an RLU value corresponding to M +2SD, performing two-point regression fitting according to the concentration-RLU value result between the zero concentration calibrator and an adjacent concentration calibrator (the adjacent concentration is 5 mu IU/mL) to obtain a linear equation, substituting the RLU value corresponding to M +2SD into the equation, and calculating to obtain a corresponding concentration, namely a lowest detection Limit (LOB).
In the following three batches of examples in tables 1-3, the lowest limit of detection (LOB) of the reagent is lower than 0.5 mu IU/mL according to the method, and the sensitivity of the reagent can reach below 0.5 mu IU/mL according to the result.
TABLE 1 results of sensitivity measurement of the kit of the present invention 1
When mannitol in reagent 1 was replaced with sorbitol and the other components in the kit were kept unchanged, the kit sensitivity according to the same method as above was 0.2597; when trehalose was replaced with sucrose in the reagent and the other components of the kit remained unchanged, the kit sensitivity was 0.2402, as shown in the following table.
TABLE 2 results of sensitivity measurement of the kit of the present invention 2
2. Repeatability test of the kit
The method comprises the steps of detecting renin samples with the concentrations of 15 mu IU/mL and 150 mu IU/mL for 10 times, respectively calculating the Coefficient of Variation (CV) of each sample, and indicating that the CV of the kit is less than 5 percent.
TABLE 3 repeatability measurements of the kits of the invention
3. Accuracy test of the kit of the invention
The detection values of the renin international standard products with the concentrations of 20, 100 and 200 mu IU/mL are respectively calculated to obtain the deviation between the detection values and the theoretical values, and the results show that the deviation of the kit for detecting the renin international standard products is less than 6 percent.
TABLE 4 repeatability measurements of the kits of the invention
4. Linear assay of the kit of the invention
A renin sample with a concentration of 500 mu IU/mL is diluted into a concentration gradient of 0.5 mu IU/mL, 5 mu IU/mL, 25 mu IU/mL, 125 mu IU/mL and 500 mu IU/mL according to a proportion, samples with each concentration are detected for 2 times, the average value of the detection results of the two times is calculated, the average value of the concentration measurement and the theoretical concentration are subjected to straight line fitting by a least square method, and a linear correlation coefficient r is calculated. The results of the three batches of experiments in the following table show that the concentration of the sample directly detectable by the reagent of the present invention is: 0.5 mu IU/mL-500 mu IU/mL.
TABLE 5 Linear measurement results of the kit of the present invention
EXAMPLE V Effect of different Components of the renin assay kit on Performance test
1. Experiment for influence of mannitol in reagent 1 on sensitivity of kit
The specific experimental scheme is similar to the sensitivity experimental scheme of the kit, the sensitivity of the reagent is obviously improved after mannitol is added into the reagent 1 according to the experimental result, the following table shows that the LOB is 1.0787 when the reagent 1 does not contain mannitol or sorbitol, and the LOB is less than 0.5 mu IU/mL in the sensitive example of the reagent.
TABLE 6 sensitivity test results in the absence of mannitol in reagent 1
According to the above method, when the concentration of mannitol in reagent 1 was changed while the other components in the kit were unchanged, the kit sensitivity data were as follows:
TABLE 7 sensitivity test results of reagent 1 containing mannitol at different concentrations
From the above results, it can be seen that the kit sensitivity is better when the mannitol concentration is 5g/L to 50 g/L. 2. Experiment of influence of buffer solution in reagent 1 on performance of kit
Under the condition that other components in the reagent 1 are unchanged and other experimental conditions are unchanged, the buffer solution, the protein and the surfactant are respectively changed, the signal value and the signal-to-noise ratio of the result of the detection calibrator are compared, and the condition with the optimal signal value and the optimal signal-to-noise ratio is screened out.
TABLE 8 results of different buffer screens for reagent 1
| Screening with different buffers | PBS | Tris | MES | HEPES |
| Calibrator (mu IU/mL) | RLU | RLU | RLU | RLU |
| 0 | 241 | 229 | 253 | 257 |
| 5.0 | 2087 | 1038 | 424 | 1370 |
| 10.0 | 4616 | 2282 | 702 | 2850 |
| 20.0 | 10430 | 6200 | 2153 | 7585 |
| 50.0 | 31840 | 16751 | 6573 | 21975 |
| 125.0 | 93595 | 50300 | 15063 | 61606 |
| 500.0 | 401078 | 211071 | 68088 | 271003 |
From the experimental result surface, the reagent 1 is selected from PBS buffer solution compared with Tris, MES and HEPES buffer solution, and the PBS buffer solution has the best effect in terms of sensitivity, signal intensity and linear relation; the pH value of the PBS buffer solution is 7.4, the pH value of the Tris buffer solution is 8.0, the pH value of the MES buffer solution is 6.0, and the pH value of the HEPES buffer solution is 7.0.
TABLE 9 results of different protein screens
From the experimental results, BSA is selected as the reagent 1 compared with Casein, and the BSA buffer solution has the best effect in terms of sensitivity, signal intensity and linear relation; the concentration of each protein used was 10 g/L.
TABLE 10 results of different surfactant screens
| Screening of different surfactants | TritonX-100 | Tween-20 | Brij35 |
| Calibrator (mu IU/mL) | RLU | RLU | RLU |
| 0 | 230 | 683 | 996 |
| 5.0 | 2176 | 2122 | 2902 |
| 10.0 | 4398 | 4558 | 3770 |
| 20.0 | 11419 | 9074 | 10710 |
| 50.0 | 33985 | 34404 | 29578 |
| 125.0 | 87199 | 71681 | 84008 |
| 500.0 | 409069 | 356313 | 346335 |
From the experimental result surface, the reagent 1 selects the optimal TritonX-100 surface activity from the aspects of sensitivity, signal intensity and linear relation, the optimal TritonX-100 concentration value is 0.1mL/L and the range is 0.05 mL/L-0.2 mL/L; the concentration of the surfactant used was 0.1mL/L
TABLE 11 results of screening for different PBS buffer concentrations
| Concentration of PBS buffer | 1mM | 5mM | 20mM | 100mM | 200mM |
| Calibrator (mu IU/mL) | RLU | RLU | RLU | RLU | RLU |
| 0 | 375 | 221 | 258 | 187 | 237 |
| 5.0 | 550 | 1495 | 1990 | 1233 | 344 |
| 10.0 | 892 | 3114 | 4569 | 2445 | 982 |
| 20.0 | 2519 | 7392 | 11397 | 5184 | 2312 |
| 50.0 | 8453 | 25292 | 28796 | 18347 | 6500 |
| 125.0 | 23277 | 62919 | 100030 | 48338 | 14921 |
| 500.0 | 141979 | 339443 | 427831 | 233750 | 63732 |
As shown by the above experimental results, the concentration of PBS buffer used in reagent 1 is optimally 20mM, preferably 5 mM-100 mM, in terms of sensitivity, signal intensity and linearity, and the pH of PBS buffer used is 7.4.
3. Effect of BSA concentration in reagent 1 on reagent Performance
And (3) comparing the signal value and the signal-to-noise ratio of the result of the detection calibrator when the reagent 1 contains BSA with different concentrations under the condition that the components of the reagent 1 are unchanged, and screening out the condition that the signal value and the signal-to-noise ratio are optimal.
TABLE 12 results of different BSA concentration screens
| BSA concentration | 0.5g/L | 1g/L | 10g/L | 50g/L | 100g/L |
| Calibrator (mu IU/mL) | RLU | RLU | RLU | RLU | RLU |
| 0 | 1065 | 427 | 231 | 243 | 310 |
| 5.0 | 1886 | 2335 | 1922 | 1342 | 711 |
| 10.0 | 3715 | 4446 | 4557 | 2888 | 1028 |
| 20.0 | 7773 | 10170 | 8897 | 7067 | 1539 |
| 50.0 | 15685 | 31816 | 27094 | 22355 | 5482 |
| 125.0 | 52959 | 74549 | 83290 | 56847 | 21771 |
| 500.0 | 271556 | 421168 | 399411 | 318325 | 134397 |
As shown by the above experimental results, the concentration of BSA in the reagent 1 is preferably 10g/L, more preferably 1 to 50g/L, in terms of sensitivity, signal intensity and linearity. 4. Experiment for influence of TritonX-100 concentration in reagent 1 on reagent performance
And (3) comparing the signal value and the signal-to-noise ratio of the result of the detection calibrator when the reagent 1 contains TritonX-100 with different concentrations under the condition that the components of the reagent 1 are unchanged, and screening out the condition that the signal value and the signal-to-noise ratio are optimal.
TABLE 13 results of screening for different TritonX-100 concentrations
| TritonX-100 concentration | 0.025mL/L | 0.05mL/L | 0.1mL/L | 0.2mL/L | 0.4mL/L |
| Calibrator (mu IU/mL) | RLU | RLU | RLU | RLU | RLU |
| 0 | 2124 | 537 | 273 | 247 | 354 |
| 5.0 | 2929 | 2699 | 2157 | 1418 | 754 |
| 10.0 | 4215 | 4715 | 4938 | 2741 | 1270 |
| 20.0 | 8572 | 11133 | 8892 | 6806 | 3241 |
| 50.0 | 20320 | 33446 | 28026 | 22450 | 8437 |
| 125.0 | 59413 | 79868 | 96250 | 56267 | 33162 |
| 500.0 | 344299 | 444621 | 408520 | 307323 | 142572 |
From the above experimental results, it was revealed that the concentration of TritonX-100 in reagent 1 is 0.1mL/L, preferably 0.05 to 0.2mL/L, in terms of sensitivity, signal intensity and linearity.
5. Experiment of influence of trehalose in reagent 2 on reagent sensitivity
The protocol was similar to the sensitivity test protocol described above, with the following table showing that reagent 2 had an LOB of 1.5864 when it contained no trehalose or sucrose, and in the reagent sensitive examples described above, the LOB was less than 0.5. mu. IU/mL; according to the experimental result, the sensitivity of the reagent 2 added with trehalose is obviously improved.
TABLE 14 sensitivity test results without trehalose
According to the above method, when the concentration of trehalose in reagent 2 was changed while the other components in the kit were unchanged, the kit sensitivity data were as follows:
TABLE 15 results of sensitivity measurements with different concentrations of trehalose
From the above results, it can be seen that the kit sensitivity is better when the trehalose concentration is 5g/L to 50 g/L.
It is to be understood that the invention disclosed is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
Claims (6)
1. A renin chemiluminescence detection kit, which is characterized in that: the kit comprises a reagent 1 and a reagent 2, wherein the reagent 1 comprises a buffer solution, 0.2-0.6 mg/mL magnetic particles, 4-12 mu g/mL biotin-labeled anti-renin monoclonal antibody and 5-50 g/L mannitol or sorbitol; and the reagent 2 comprises buffer solution, 2.0-6.0 mu g/mL acridinium ester labeled anti-renin monoclonal antibody and 5-50 g/L trehalose or sucrose;
the buffer solution is PBS buffer solution with pH6.0-8.0 and 5 mM-100 mM;
the reagent kit 1 also comprises 1 g/L-50 g/L bovine serum albumin, 5 mL/L-20 mL/L glycerol, 0.05 mL/L-0.2 mL/L TritonX-100, 10 mg/L-50 mg/L4-aminoantipyrine and 0.5 mL/L-5 mL/L proclin-300;
the reagent kit 2 also comprises 5-20 g/L casein, 5-20 mL/L glycerol, 0.05-0.2 mL/L TritonX-100, 0.5-5 mL/L proclin-300 and 10-50 mg/L4-aminoantipyrine.
2. The renin chemiluminescent assay kit according to claim 1, said reagent 1 comprising 0.4mg/mL magnetic microparticles, 8 μ g/mL biotin-labeled anti-renin monoclonal antibody, 20mM ph7.4pbs buffer solution, 10g/L bovine serum albumin, 20g/L mannitol or sorbitol, 10mL/L glycerol, 0.1mL/L triton x-100, 20 mg/L4-aminoantipyrine and 1mL/L proclin-300.
3. The renin chemiluminescent assay kit according to claim 1, said reagent 2 comprising 4.0 μ g/mL acridinium ester labeled anti-renin monoclonal antibody, 20mM ph7.4PBS buffer solution, 10g/L casein, 10g/L trehalose or sucrose, 10mL/L glycerol, 0.1mL/L triton x-100, 1mL/L proclin-300 and 20 mg/L4-aminoantipyrine.
4. The renin chemiluminescent assay kit according to any one of claims 1 to 3 further comprising a calibrator comprising PBS buffer, renin antigen, casein, bovine serum albumin, trehalose or sucrose, glycerol, TritonX-100, proclin-300 and 4-aminoantipyrine; the calibrator comprises 10 mu IU/mL or 100 mu IU/mL of renin antigen, 5 mM-100 mM, PBS buffer solution with pH of 6.0-8.0, 5 g/L-20 g/L of casein, 20 g/L-100 g/L of bovine serum albumin, 5 g/L-50 g/L of trehalose or sucrose, 5 mL/L-20 mL/L of glycerol, 0.05 mL/L-0.2 mL/L of TritonX-100, 0.5 mL/L-5 mL/L of proclin-300 and 10 mg/L-50 mg/L of 4-aminoantipyrine.
5. The renin chemiluminescent assay kit of claim 4, the calibrator comprises 10 μ IU/mL or 100 μ IU/mL renin antigen, 20mM ph7.4PBS buffer, 10g/L casein, 50g/L bovine serum albumin, 20g/L trehalose or sucrose, 10mL/L glycerol, 0.1mL/L triton x-100, 1mL/L proclin-300 and 20 mg/L4-aminoantipyrine.
6. The renin chemiluminescent assay kit according to any one of claims 1 to 3 further comprising a chemiluminescent substrate solution comprising solution A and solution B; the A solution is H2O2And the solution B is NaOH solution.
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